Expanding the genetic spectrum of TUBB1-related thrombocytopenia

β1-tubulin plays a major role in proplatelet formation and platelet shape maintenance, and pathogenic variants in TUBB1 lead to thrombocytopenia and platelet anisocytosis (TUBB1-RT). To date, the reported number of pedigrees with TUBB1-RT and of rare TUBB1 variants with experimental demonstration of pathogenicity is limited. Here, we report 9 unrelated families presenting with thrombocytopenia carrying six β1-tubulin variants: p.Cys12Leufs12*, p.Thr107Pro, p.Gln423*, p.Arg359Trp, p.Gly109Glu, and p.Gly269Asp, the last of which novel. Segregation studies showed incomplete penetrance of these variants for platelet traits. Indeed, most carriers showed macrothrombocytopenia, some only increased platelet size and a minority no abnormalities. Moreover, only homozygous carriers of the p.Gly109Glu variant, displayed macrothrombocytopenia, highlighting the importance of allele burden in the phenotypic expression of TUBB1-RT. The p.Arg359Trp, p.Gly269Asp and p.Gly109Glu variants deranged β1-tubulin incorporation into the microtubular marginal ring in platelets, while had negligible effect on platelet activation, secretion or spreading, suggesting that β1-tubulin is dispensable for these processes. Transfection of TUBB1 missense variants in CHO cells altered β1-tubulin incorporation into the microtubular network. In addition, TUBB1 variants markedly impaired proplatelet formation from peripheral blood CD34+ cell-derived megakaryocytes. Our study, using in vitro modeling, molecular characterization, and clinical investigations provides a deeper insight into the pathogenicity of rare TUBB1 variants. These novel data expand the genetic spectrum of TUBB1-RT and highlight a remarkable heterogeneity in its clinical presentation, indicating that allelic burden or combination with other genetic or environmental factors modulate the phenotypic impact of rare TUBB1 variants.

Mechanical counterbalance of kinesin and dynein motors in microtubular network regulates cell mechanics, 3D architecture, and mechanosensing

Microtubules (MTs) and MT motor proteins form active 3D networks made of unstretchable cables with rod-like bending mechanics that provide cells with a dynamically changing structural scaffold. In this study, we report an antagonistic mechanical balance within the dynein-kinesin microtubular motor system. Dynein activity drives microtubular network inward compaction, while isolated activity of kinesins bundles and expands MTs into giant circular bands that deform the cell cortex into discoids. Furthermore, we show that dyneins recruit MTs to sites of cell adhesion increasing topographic contact guidance of cells, while kinesins antagonize it via retraction of MTs from sites of cell adhesion. Actin-to-microtubules translocation of septin-9 enhances kinesins-MTs interactions, outbalances activity of kinesins over dyneins and induces discoid architecture of cells. These orthogonal mechanisms of MT network reorganization highlight the existence of an intricate mechanical balance between motor activities of kinesins and dyneins that controls cell 3D architecture, mechanics, and cell-microenvironment interactions.

A scallop theorem for cells moving in 3D

The famous scallop theorem proposed by Purcell in 1977 states that self-propelled objects swimming at low Reynolds number must follow a cycle of shape changes that breaks temporal symmetry. This should hold true for crawling cells as well. However a clear mechanism for this symmetry breaking is still elusive. Here we show that cells embedded in 3D matrix form at both sides of the nucleus force dipoles driven by myosin that locally and periodically pinch the matrix. Using a combination of 3D live cell imaging, traction force microscopy and a minimal model with multipolar expansion, we show that the existence of a phase shift between the two dipoles involves mainly the microtubular network and is required for directed cell motion. We confirm this mechanism by triggering local dipolar contractions with a laser, which leads to directed motion. Our study reveals that the cell controls its motility by synchronising dipolar forces distributed at front and back. This result opens new strategies to externally control cell motion.

Microtubules regulate cardiomyocyte transversal Young’s modulus

The field of cardiomyocyte mechanobiology is gaining significant attention, due to accumulating evidence concerning the significant role of cellular mechanical effects on the integrated function of the heart. To date, the protein titin has been demonstrated as a major contributor to the cardiomyocytes Young’s modulus (YM). The microtubular network represents another potential regulator of cardiac mechanics. However, the contribution of microtubules (MTs) to the membrane YM is still understudied and has not been interrogated in the context of myocardial infarction (MI) or mechanical loading and unloading. Using nanoscale mechanoscanning ion conductance microscopy, we demonstrate that MTs contribute to cardiomyocyte transverse YM in healthy and pathological states with different mechanical loading. Specifically, we show that posttranslational modifications of MTs have differing effects on cardiomyocyte YM: Acetylation provides flexibility, whereas detyrosination imparts rigidity. Further studies demonstrate that there is no correlation between the total protein amount of acetylated and detyrosinated MT. Yet, in the polymerized-only populations, an increased level of acetylation results in a decline of detyrosinated MTs in an MI model.

KIF1-binding protein interacts with KIF3A in haploid male germ cells

Male fertility relies on the production of functional spermatozoa. Spermatogenesis is a complex differentiation process that is characterized by meiosis and dramatic morphogenesis of haploid cells. Spermatogenesis involves active changes in the microtubular network to support meiotic divisions, cell polarization, the reshaping of the nucleus, and the formation of a flagellum. Previously, we have demonstrated that a microtubule-based anterograde transport motor protein KIF3A is required for the sperm tail formation and nuclear shaping during spermatogenesis. In this study, we show that KIF3A interacts with a KIF1-binding protein (KBP) in the mouse testis. We have characterized the expression and localization pattern of KBP during spermatogenesis and localized both KIF3A and KBP in the cytoplasm of round spermatids and manchette of elongating spermatids. Interestingly, KBP localized also in the late chromatoid body (CB) of elongating spermatids, whose function involves intracellular movement and association with the microtubular network. Altogether our results suggest a role for KBP in spermatid elongation and in the function of the late CB.

The Involvement of Microtubules and Actin during the Infection of Japanese Encephalitis Virus in Neuroblastoma Cell Line, IMR32

The role of the cytoskeleton, actin, and microtubules were examined during the process of Japanese encephalitis (JEV) infection in a human neuroblastoma cell line, IMR32. Cytochalasin D and nocodazole were used to depolymerise the cellular actin and microtubules, respectively, in order to study the effect of JEV infection in the cell. This study shows that depolymerisation of the actin cytoskeleton at early process of infection inhibits JEV infection in the cell; however infection was not inhibited when depolymerisation occurred at the later stage of infection. The microtubules, on the other hand, are required at 2 points in infection. The antigen production in the cells was inhibited when the infected cells were treated at time up to 2 hours after inoculation and there was no significant effect at later times, while the viable virus released continued to be affected until 10 hours after inoculation. In conclusion, infection of JEV in IMR32 cells required actin to facilitate early process in infection and the microtubular network is utilised as the transport system to the virus replication site and the release of mature virus.